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A METHOD FOR IDENTIFYING RIBOSOME PAUSE SITES IN MESSENGER RNA THROUGH NEW SEQUENCING TECHNOLOGY

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title
A METHOD FOR IDENTIFYING RIBOSOME PAUSE SITES IN MESSENGER RNA THROUGH NEW SEQUENCING TECHNOLOGY
author
Bender, R. Hugh F.
abstract
Ribosome frameshifting, pausing, and termination events have a profound effect on the accuracy and reliability of protein synthesis. A variety of translational control mechanisms combine to ensure the fidelity of this process. Mechanisms, such as aminoacyl-tRNA selection and codon:anticodon interactions, are well characterized. However, more complex mechanisms, such as mRNA secondary structures, require a new approach for further study. We suggest that these mechanisms may be studied on a global scale by mapping the distribution of ribosomes along a select group of mRNAs. A combination of techniques for ribosome-protected mRNA fragment isolation (ribosome footprinting) and high-throughput sequencing—a method for simultaneously sequencing up to 40 million fragments—provides the tools for studying global translational control mechanisms. We plan to sequence a set of ribosome footprints using Illumina’s Genome Analyzer, which requires the presence of two unique adapter sequences on either end of these fragments. Conventional methods for adding these adapter sequences to RNA fragments result in significant material loss. Here we propose an improved method for isolating and converting small RNA fragments to a sequencing-ready form without the use of gel purification or sample amplification (PCR) steps. We also critique a similar method published by Ingolia, et al., (2009).
subject
Translation
Protein Synthesis
Ribosomes
High-throughput Sequencing
contributor
Tague, Brian (committee chair)
Curran, James (committee member)
Fetrow, Jacquelyn (committee member)
Ornelles, David (committee member)
date
2009-08-03T14:30:12Z (accessioned)
2010-06-18T18:59:28Z (accessioned)
2009-08-03T14:30:12Z (available)
2010-06-18T18:59:28Z (available)
2009-08-03T14:30:12Z (issued)
degree
Biology (discipline)
identifier
http://hdl.handle.net/10339/14857 (uri)
language
en_US (iso)
publisher
Wake Forest University
rights
Release the entire work for access only to the Wake Forest University system for one year from the date below. After one year, release the entire work for access worldwide. (accessRights)
type
Thesis

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